Novel inhibitors of the Plasmodium falciparum electron transport chain.

Due to an increased need for new antimalarial chemotherapies that show potency against Plasmodium falciparum, researchers are targeting new processes within the parasite in an effort to circumvent or delay the onset of drug resistance. One such promising area for antimalarial drug development has been the parasite mitochondrial electron transport chain (ETC). Efforts have been focused on targeting key processes along the parasite ETC specifically the dihydroorotate dehydrogenase (DHOD) enzyme, the cytochrome bc 1 enzyme and the NADH type II oxidoreductase (PfNDH2) pathway. This review summarizes the most recent efforts in antimalarial drug development reported in the literature and describes the evolution of these compounds.

The plasma membrane of microaerophilic protists: oxidative and nitrosative stress.

The trans-plasma-membrane electrochemical potential of microaerophilic protists was monitored by the use of voltage-sensitive charged lipophilic fluorophores; of the many available probes, the anionic oxonol dye bis(1,3-dibarbituric acid)-trimethine oxonol [DiBAC(4)(3)] is an example of one which has been successfully employed using fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. Several microaerophilic protists have been investigated with this dye; these were Giardia intestinalis, Trichomonas vaginalis, Tritrichomonas foetus, Hexamita inflata and Mastigamoeba punctachora. Under conditions where they exhibit normal vitality, these organisms exclude DiBAC(4)(3) by virtue of their maintenance of a plasma-membrane potential (negative inside). Uptake of the fluorophore is indicative of disturbance to this membrane (i.e. by inhibition of pump/leak balance, blockage of channels or generation of ionic leaks), and is indicative of metabolic perturbation or environmental stress. Here, it is shown that oxidative or nitrosative stress depolarizes the plasma membranes of the aforementioned O(2)-sensitive organisms and allows DiBAC(4)(3) influx. Oxonol uptake thereby provides a sensitive and early indication of plasma-membrane perturbation by agents that may lead to cytotoxicity and eventually to cell death by necrotic or apoptotic pathways.

Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay.

AIMS: To examine A, C, Y, and W135 Neisseria meningitidis serogroup characterisation by ultrasonic standing wave enhanced latex agglutination tests (USELATs) of clinical samples. In addition, to determine USELAT enhancement of detection sensitivity for the individual antigens compared with conventional card latex agglutination tests (LATs). METHODS: Wellcogen (Abbott Murex), Slidex meningite kit 5 (bioMerieux), and Pastorex (Sanofi) kits and beads coated in house with antibodies to Y and to W135 alone were tested. Positive control antigens consisted of A and C polysaccharide preparations and the Pastorex Y/W135 kit sample. The limiting concentrations of antigen detection were determined by USELAT and by LAT. Thirty five clinical samples (plasma), previously characterised by the polymerase chain reaction (PCR) and culture, were tested by USELAT and, when sample volume allowed, by LAT. RESULTS: USELAT enhancement of control antigen detection ranged from 16 to 128 fold for the different latex systems. Enhancements for the different control antigens were comparable between kits. USELAT tests of clinical (A/C/Y/W135) samples (n = 15) with the Wellcogen (A/C/Y/W135) and Slidex meningite (A/C/Y/W135) kits showed comparable specificities. A set (n = 22) of Y and W135 samples gave 18, 19, and 17 positive results for Wellcogen (A/C/Y/W135), Pastorex (A/C/Y/W135), and in house beads (Y/W135), respectively. Positive USELAT PCR and culture results were concordant. A typical sensitivity for the commercial kits was 80% (Wellcogen). CONCLUSIONS: USELAT identified serogroups for 80% of samples, whereas LATs identified only 40%. The USELAT detection of the A, C, Y, and W135 antigen serogroups showed comparable enhancement for the kits tested. The commercial availability of latex beads coated with antibody to the Y and W135 serogroups would expedite their identification.

The antioxidant potential of pyruvate in the amitochondriate diplomonads Giardia intestinalis and Hexamita inflata.

Giardia intestinalis and Hexamita inflata are microaerophilic protozoa which rely on fermentative metabolism for energy generation. These organisms have developed a number of antioxidant defence strategies to cope with elevated O(2) tensions which are inimical to survival. In this study, the ability of pyruvate, a central component of their energy metabolism, to act as a physiological antioxidant was investigated. The intracellular pools of 2-oxo acids in G. intestinalis were determined by HPLC. With the aid of a dichlorodihydrofluorescein diacetate-based assay, intracellular reactive oxygen species generation by G. intestinalis and H. inflata suspensions was monitored on-line. Addition of physiologically relevant concentrations of pyruvate to G. intestinalis and H. inflata cell suspensions was shown to attenuate the rate of H(2)O(2)- and menadione-induced generation of reactive oxygen species. In addition, pyruvate was also shown to decrease the generation of low-level chemiluminescence arising from the oxygenation of anaerobic suspensions of H. inflata. In contrast, addition of pyruvate to suspensions of respiring Saccharomyces cerevisiae was shown to increase the generation of reactive oxygen species. These data suggest that (i) in G. intestinalis and H. inflata, pyruvate exerts antioxidant activity at physiological levels, and (ii) it is the absence of a respiratory chain in the diplomonads which facilitates the observed antioxidant activity.

Na(+)-dependent pH regulation by the amitochondriate protozoan parasite Giardia intestinalis.

Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques ((31)P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show that G. intestinalis trophozoites exposed to an extracellular pH range of 6.0--7.5 maintain their cytosolic pH (pH(i)) within the range 6.7--7.1. Maintenance of the resting pH(i) was Na(+)-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH(i) from an intracellular acidosis was also Na(+)-dependent, with the rate of recovery varying with the extracellular Na(+) concentration in a saturable manner (K(m) = 18 mm; V(max) = 10 mm H(+) min(-1)). The recovery of pH(i) from an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na(+)/H(+) exchanger(s) in the regulation of pH(i) in G. intestinalis is discussed.

The membrane potential of Giardia intestinalis.

Giardia intestinalis is a primitive microaerophilic protozoan parasite which colonises the upper intestine of humans. Despite the evolutionary and medical significance of this organism, its physiology is very poorly understood. In this study we have used a novel flow cytometric technique to make quantitative measurements of the electrical potential across the plasma membrane of G. intestinalis trophozoites. In media lacking both K(+) and Na(+), G. intestinalis trophozoites maintained a high negative plasma membrane potential (Psi(m)) of -134+/-3 mV. The Psi(m) was unaffected by the addition of Na(+) to the extracellular medium, whereas the addition of K(+) resulted in a significant membrane depolarisation, consistent with the G. intestinalis trophozoite plasma membrane having a significant (electrophoretic) permeability to K(+). The membrane was also depolarised by the H(+) ionophore m-chlorophenylhydrazone and by the H(+) ATPase inhibitors dicyclohexylcarbodiimide and N-ethylmaleimide. These results are consistent with G. intestinalis trophozoites maintaining a high resting Psi(m), originating at least in part from an electrogenic H(+) pump acting in concert with a K(+) diffusion pathway.

Carbohydrate and Amino Acid Fermentation in the Free-Living Primitive Protozoon Hexamita sp.

Hexamita sp. is an amitochondriate free-living diplomonad which inhabits O(2)-limited environments, such as the deep waters and sediments of lakes and marine basins. C nuclear magnetic resonance spectroscopy reveals ethanol, lactate, acetate, and alanine as products of glucose fermentation under microaerobic conditions (23 to 34 muM O(2)). Propionic acid and butyric acid were also detected and are believed to be the result of fermentation of alternative substrates. Production of organic acids was greatest under microaerobic conditions (15 muM O(2)) and decreased under anaerobic (<0.25 muM O(2)) and aerobic (200 to 250 muM O(2)) conditions. Microaerobic incubation resulted in the production of high levels of oxidized end products (70% acetate) compared to that produced under anoxic conditions (20% acetate). In addition, data suggest that Hexamita cells contain the arginine dihydrolase pathway, generating energy from the catabolism of arginine to citrulline, ornithine, NH(4), and CO(2). The rate of arginine catabolism was higher under anoxic conditions than under microaerobic conditions. Hexamita cells were able to grow in the absence of a carbohydrate source, albeit with a lower growth rate and yield.

Evolution of the hydrogenosome.

Since its discovery almost 25 years ago the enigmatic hydrogenosome, a redox organelle of anaerobic unicellular eukaryotes, has puzzled evolutionists as to its origin and function. Synthesis of recent molecular, physiological and morphological studies now favours the hypothesis that hydrogenosomes derived from a modification of pre-existing mitochondria, and argues against the previously held view that the hydrogenosome had a polyphyletic origin. These data provide evidence for a more ancient origin of mitochondria than hitherto thought.

Oxygen uptake and antioxidant responses of the free-living diplomonad Hexamita sp.

The free-living anaerobic flagellate Hexamita sp. was observed to actively consume O2 with a K(m) O2 of 13 microM. Oxygen consumption increased linearly with O2 tension up to a threshold level of 100 microM, above which it was inhibited. Oxygen uptake was supported by a number of substrates but probably not coupled to energy conservation as cytochromes could not be detected spectro-photometrically. In addition, inhibitors specific for respiratory chain components did not significantly affect O2 uptake. Respiration was however, partially inhibited by flavoprotein and iron-sulfur protein inhibitors. NAD(P)H supported O2 consumption was measured in both particulate and soluble fractions; this activity was partially inhibited by quinacrine. A chemosensory response was observed in cells exposed to air, however no response was observed in the presence of superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase activity was present. Superoxide dismutase was sensitive to NaN3, and H2O2 but not KCN, suggesting a Fe prosthetic group. Flow cytometric analysis revealed that thiol levels in live cells were depleted in the presence of t-butyl H2O2. The observed NADPH-driven glutathione reductase activity is believed to recycle oxidized thiols in order to re-establish reduced thiol levels in the cell. The corresponding thiol cycling enzyme glutathione peroxidase could not be detected. The ability to withstand high O2 tensions (100 microM) would enable Hexamita to spend short periods in a wider range of habitats. Prolonged exposure to O2 tensions higher than 100 microM leads to irreversible damage and cell death.